Endothelin receptor antagonists

ABSTRACT

Novel isooxazoles, oxazoles, thiazoles, isothiazoles and imidazoles, pharmaceutical compositions containing these compounds and their use as endothelin receptor antagonists are described.

This application claims the benefit of Provisional application Ser. No.60/010,992 filed Feb. 1, 1996.

FIELD OF THE INVENTION

The present invention relates to isooxazoles, oxazoles, thiazoles,isothiazoles and imidazoles, pharmaceutical compositions containingthese compounds and their use as endothelin receptor antagonists.

Endothelin (ET) is a highly potent vasoconstrictor peptide synthesizedand released by the vascular endothelium. Endothelin exists as threeisoforms, ET-1, ET-2 and ET-3. [Unless otherwise stated "endothelin"shall means any or all of the isoforms of endothelin]. Endothelin hasprofound effects of the cardiovascular system, and in particular, thecoronary, renal and cerebral circulation. Elevated or abnormal releaseof endothelin is associated with smooth muscle contraction which isinvolved in the pathogenesis of cardiovascular, cerebrovascular,respiratory and renal pathophysiology. Elevated levels of endothelinhave been reported in plasma from patients with essential hypertension,acute myocardial infarction, subarachnoid hemorrhage, atherosclerosis,and patients with uraemia undergoing dialysis.

In vivo, endothelin has pronounced effects on blood pressure and cardiacoutput. An intravenous bolus injection of ET (0.1 to 3 nmol/kg) in ratscauses a transient, dose-related depressor response (lasting 0.5 to 2minutes) followed by a sustained, dose-dependent rise in arterial bloodpressure which can remain elevated for 2 to 3 hours following dosing.Doses above 3 nmol/kg in a rat often prove fatal.

Endothelin appears to produce a preferential effect in the renalvascular bed. It produces a marked, long-lasting decrease in renal bloodflow, accompanied by a significant decrease in GFR, urine volume,urinary sodium and potassium excretion. Endothelin produces a sustainedantinatriuretic effect, despite significant elevations in atrialnatriuretic peptide. Endothelin also stimulates plasma renin activity.These findings suggest that ET is involved in the regulation of renalfunction and is involved in a variety of renal disorders including acuterenal failure, cyclosporine nephrotoxicity, radio contrast induced renalfailure and chronic renal failure.

Studies have shown that in vivo, the cerebral vasculature is highlysensitive to both the vasodilator and vasoconstrictor effects ofendothelin. Therefore, ET may be an important mediator of cerebralvasospasm, a frequent and often fatal consequent of subarachnoidhemorrhage.

ET also exhibits direct central nervous system effects such as severeapnea and ischemic lesions which suggests that ET may contribute to thedevelopment of cerebral infarcts and neuronal death.

ET has also been implicated in myocardial ischemia (Nichols et al. Br.J. Pharm. 99: 597-601, 1989 and Clozel and Clozel, Circ. Res., 65:1193-1200, 1989) coronary vasospasm (Fukuda et al., Eur. J. Pharm. 165:301-304, 1989 and Luscher, Circ. 83: 701, 1991) heart failure,proliferation of vascular smooth muscle cells, (Takagi, Biochem &Biophys, Res. Commun.: 168: 537-543, 1990, Bobek et al., Am. J. Physiol.258:408-C415, 1990) and atherosclerosis, (Nakaki et al., Biochem. &Biophys. Res. Commun. 158: 880-881, 1989, and Lerman et al., New Eng. J.of Med. 325: 997-1001, 1991). Increased levels of endothelin have beenshown after coronary balloon angioplasty (Kadel et al., No. 2491 Circl.82: 627, 1990).

Further, endothelin has been found to be a potent constrictor ofisolated mammalian airway tissue including human bronchus (Uchida etal., Eur. J. of Pharm. 154: 227-228 1988, LaGente, Clin. Exp. Allergy20: 343-348, 1990; and Springall et al., Lancet, 337: 697-701, 1991).Endothelin may play a role in the pathogenesis of interstitial pulmonaryfibrosis and associated pulmonary hypertension, Glard et al., ThirdInternational Conference on Endothelin, 1993, p. 34 and ARDS (AdultRespiratory Distress Syndrome), Sanai et al., Supra, p. 112.

Endothelin has been associated with the induction of hemorrhagic andnecrotic damage in the gastric mucose (Whittle et al., Br. J. Pharm. 95:1011-1013, 1988); Raynaud's phenomenon, Cinniniello et al., Lancet 337:114-115, 1991); Crohn's Disease and ulcerative colitis, Munch et al.,Lancet, Vol. 339, p. 381; Migraine (Edmeads, Headache, February 1991 p127); Sepsis (Weitzberg et al., Circ. Shock 33: 222-227, 1991; Pittet etal., Ann. Surg. 213: 262-264, 1991), Cyclosporin-inducted renal failureor hypertension (Eur. J. Pharmacol., 180: 191-192, 1990 Kidney Int, 37:1487-1491, 1990) and endotoxin shock and other endotoxin induceddiseases (Biochem, Biophys. Res. Commun., 161: 1220-1227, 1989, ActaPhysiol. Scand. 137: 317-318, 1989) and inflammatory skin diseases,(Clin Res. 41: 451 and 484, 1993).

Endothelin has also been implicated in preclampsia of pregnancy. Clarket al., Am. J. Obstet. Gynecol. March 1992, p. 962-968; Kamor et al., N.Eng. J. of Med., Nov. 22, 1990, p. 1486-1487; Dekker et al., Eur J. Ob.and Gyn. and Rep. Bio. 40 (1991) 215-220; Schiff et al., Am. J. Ostet.Gynecol. February 1992, p. 624-628; diabetes mellitus, Takahashi et al.,Diabetologia (1990) 33: 306-310; and acute vascular rejection followingkidney transplant, Watschinger et al., Transplantation Vol. 52, No. 4,pp. 743-746.

Endothelin stimulates both bone resorption and anabolism and may have arole in the coupling of bone remodeling. Tatrai et al. Endocrinolgy,Vol. 131, p. 603-607.

Endothelin has been reported to stimulate the transport of sperm in theuterine cavity, Casey et al., J. Clin. Endo and Metabolsim, Vol. 74, No.1, pp. 223-225, therefore endothelin antagonists may be useful as malecontraceptives. Endothelin modulates the ovarian/menstrual cycle,Kenegsberg, J. of Clin. Endo. and Met., Vol. 74, No. 1, p. 12, and mayalso play a role in the regulation of penile vascular tone in man, Lauet al., Asia Pacific J. of Pharm., 1991, 6:287-292 and Tejada et al., J.Amer. Physio. Soc. 1991, H1078-H1085. Endothelin also mediates a potentcontraction of human prostatic smooth muscle, Langenstroer et al., J.Urology, Vol. 149, p. 495-499.

Thus, endothelin receptor antagonists would offer a unique approachtoward the pharmacotherapy of hypertension, renal failure, ishcemiainduced renal failure, sepsis-endotoxin induced renal failure,prophylaxis and/or treatment of radio-contrast induced renal failure,acute and chronic cyclosporin induced renal failure, cerebrovasculardisease, myocardial ischemia, angina, heart failure, asthma, pulmonaryhypertension, pulmonary hypertension secondary to intrinsic pulmonarydisease, atheroschlerosis, Raynaud's phenomenon, ulcers, sepsis,migraine, glaucoma, endotoxin shock, endotoxin induced multiple organfailure or disseminated intravascular coagulation, cyclosporin-inducedrenal failure and as an adjunct in angioplasty for prevention ofrestenosis, diabetes, preclampsia of pregnancy, bone remodeling, kidneytransplant, male contraceptives, infertility and priaprism and benignprostatic hypertrophy.

SUMMARY OF THE INVENTION

This invention comprises compounds represented by Formula (I) andpharmaceutical compositions containing these compounds, and their use asendothelin receptor antagonists which are useful in the treatment of avariety of cardiovascular and renal diseases including but not limitedto: hypertension, acute and chronic renal failure, cyclosporine inducednephrotoxicity, benign prostatic hypertrophy, pulmonary hypertension,migraine, stroke, cerebrovascular vasospasm, myocardial ischemia,angina, heart failure, atherosclerosis, and as an adjunct in angioplastyfor prevention of restenosis.

This invention further constitutes a method for antagonizing endothelinreceptors in an animal, including humans, which comprises administeringto an animal in need thereof an effective amount of a compound ofFormula (I).

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are represented by structural Formula(I): ##STR1## wherein Z is ##STR2## D is O or S; E is O, S or NR₁₅ ;

P is tetrazol-5-yl, CO₂ R₆ or C(O)N(R₆)S(O)_(q) R₁₀ ;

R^(a) is hydrogen or C₁₋₆ alkyl;

R₁ is independently hydrogen, Ar or C₁₋₆ alkyl;

R₂ is Ar, C₁₋₈ alkyl, C(O)R₁₄ or ##STR3## R₃ and R₅ are independentlyR₁₃ OH, C₁₋₈ alkoxy, S(O)_(q) R₁₁, N(R₆)₂, Br, F, I, Cl, CF₃, NHCOR₆ R₁₃CO₂ R₇, --X--R₉ --Y or --X(CH₂)_(n) R₈ wherein each methylene groupwithin --X(CH₂)_(n) R₈ may be unsubstituted or substituted by one or two--(CH₂)_(n) Ar groups;

R₄ is independently R₁₁, OH, C₁₋₅ alkoxy, S(O)_(q) R₁₁, N(R₆)₂, Br, F,I, Cl or NHCOR₆, wherein the C₁₋₅ alkoxy may be unsubstituted orsubstituted by OH, methoxy or halogen,

R₆ is independently hydrogen or C₁₋₈ alkyl;

R₇ is independently hydrogen, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₈alkynyl, all of which may be unsubstituted or substituted by one or moreOH, N(R₆)₂, CO₂ R₁₂, halogen or XC₁₋₁₀ alkyl; or R₇ is (CH₂)_(n) Ar;

R₈ is independently R₁₁, CO₂ R₇, CO₂ C(R₁₁)₂ O(CO)XR₇, PO₃ (R₇)₂, SO₂NR₇ R₁₁, NR₇ SO₂ R₁₁, CONR₇ SO₂ R₁₁, SO₃ R₇, SO₂ R₇, P(O)(OR₇)R₇, CN,CO₂ (CH₂)_(m) C(O)N(R₆)₂, C(R₁₁)₂ N(R₇)₂, C(O)N(R₆)₂, NR₇ C(O)NR₇ SO₂R₁₁, tetrazole or OR₆ ;

R₉ is independently a bond, C₁₋₁₀ alkylene, C₁₋₁₀ alkenylene, C₁₋₁₀alkylidene, C₁₋₁₀ alkynylene, all of which may be linear or branched, orphenylene, all of which may be unsubstituted or substituted by one ofmore OH, N(R₆)₂, COOH or halogen;

R₁₀ is independently C₁₋₁₀ alkyl, N(R₆)₂ or Ar;

R₁₁ is independently hydrogen, Ar, C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, all of which may be unsubstituted or substituted by one or moreOH, CH₂ OH, N(R₆)₂ or halogen;

R₁₂ is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl or C₂₋₇ alkynyl;

R₁₃ is independently divalent Ar, C₁₋₁₀ alkylene, C₁₋₁₀ alkylidene,C₂₋₁₀ alkenylene, all of which may be unsubstituted or substituted byone or more OH, CH₂ OH, N(R₆)₂ or halogen;

R₁₄ is independently hydrogen, C₁₋₁₀ alkyl, XC₁₋₁₀ alkyl, Ar or XAr;

R₁₅ is independently C₁₋₆ alkyl or phenyl substituted by one or two C₁₋₆alkyl, OH, C₁₋₅ alkoxy, S(O)_(q) R₆, N(R₆)₂, Br, F, I, Cl, CF₃ or NHCOR₆;

X is independently (CH₂)_(n), O, NR₆ or S(O)_(q) ;

Y is independently CH₃ or X(CH₂)_(n) Ar;

Ar is independently: ##STR4## naphthyl, furyl, oxozolyl, indolyl,pyridyl, thienyl, oxazolidinyl, thiazolyl, isothiazolyl, pyrazolyl,triazolyl, tetrazolyl, imidazolyl, imidazolidinyl, thiazolidinyl,isoxazolyl, oxadiazolyl, thiadiazolyl, morpholinyl, piperidinyl,piperazinyl, pyrrolyl, or pyrimidyl; all of which may be unsubstitutedor substituted by one or more Z₁ or Z₂ groups

A is independently C═O, or (C(R₆)₂)_(m) ;

B is independently --CH₂ -- or --O--;

Z₁ and Z₂ are independently hydrogen, XR₆, C₁₋₈ alkyl, (CH₂)_(q) CO₂ R₆,C(O)N(R₆)₂, CN, (CH₂)_(n) OH, NO₂, F, Cl, Br, I, N(R₆)₂, NHC(O)R₆,X(CH₂)_(n) R₈, O(CH₂)_(m) C(O)NR_(a) SO₂ R₁₅, (CH₂)_(m) OC(O)NR_(a) SO₂R₁₅, O(CH₂)_(m) NR_(a) C(O)NR₁ SO₂ R₁₅, or tetrazolyl which may besubstituted or unsubstituted by C₁₋₆ alkyl, CF₃ or C(O)R₆ ;

Ar' is naphthyl, furyl, oxozolyl, indolyl, pyridyl, thienyl,oxazolidinyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, tetrazolyl,imidazolyl, imidazolidinyl, thiazolidinyl, isoxazolyl, oxadiazolyl,thiadiazolyl, morpholinyl, piperidinyl, piperazinyl, pyrrolyl, orpyrimidyl; all of which may be unsubstituted or substituted by one ormore XR₉ --Y, Z₁ or Z₂ groups;

m is independently 1 to 3;

n is independently 0 to 6;

q is independently 0, 1 or 2;

provided R₃, R₄ and R₅ are not O--O(CH₂)_(n) Ar;

or a pharmaceutically acceptable salt thereof.

All alkyl, alkenyl, alkynyl and alkoxy groups may be straight orbranched.

The compounds of the present invention may contain one or moreasymmetric carbon atoms and may exist in racemic and optically activeform. All of these compounds and diastereoisomers are contemplated to bewithin the scope of the present invention.

The preferred compounds are:

(E)-alpha-[[5-[4-[(2-carboxyphenyl)methoxy]-2-methoxypyrid-5-yl]isoxazol-4-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid;

(E)-alpha-[[5-(3-carboxy-5-chlorothien-2-yl)isoxazol-4-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid;

(E)-alpha-[[3-[4-[(2-carboxyphenyl)methoxy]-2-methoxypyrid-5-yl]isoxazol-4-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid;

(E)-alpha-[[3-(3-carboxy-5-chlorothien-2-yl)isoxazol-4-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid;

(E)-alpha-[[3-Butyl-4-[4-[(2-carboxyphenyl)methoxy]-2-methoxypyrid-5-yl]isoxazol-5-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid;

(E)-alpha-[[5-Butyl-4-[4-[(2-carboxyphenyl)methoxy]-2-methoxypyrid-5-yl]isoxazol-3-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid; ##STR5## Compounds of the Formula (Id), can be prepared startingfrom a ketone of Formula (2) wherein Ar' is defined as in formula (Id)##STR6## which is reacted with dimethyl carbonate in the presence of abase such as sodium hydride at 40-70° C. to provide a keto ester ofFormula (3). ##STR7## Reaction of keto ester of Formula (3) withN,N-dimethylformamide dimethyl acetal in hot toluene provides theenamine of Formula (4). ##STR8## Treatment of compound of Formula (4)with hydroxylamine hydrochloride (NH₂ OH.HCl) in a suitable solvent suchas either methanol or aqueous methanol and in the presence of a basesuch as sodium acetate affords a mixture isoxazoles of Formulas (5) and(6), which can be separated by silica gel chromatography or by selectiverecrystallization. ##STR9## Reduction of isoxazol of Formula (5) witheither diisobutylaluminum hydride in dichloromethane or lithiumborohydride in tetrahydrofuran at low temperature produces the alcoholof Formula (7). ##STR10## Oxidation of the primary alcohol of Formula(7) with either Jones reagent at ambient temperature or with manganesedioxide in refluxing toluene affords the aldehyde of Formula (8).##STR11## Knoevenagel condensation of isoxazolyl aldehyde of Formula (8)with a half acid of Formula (9), wherein R₁₆ is allyl and R₂ is definedas in formula (Id) ##STR12## in a solvent such as benzene at reflux, inthe presence of piperidinium acetate with azeotropic removal of waterusing a Dean-Stark apparatus provides an ester of Formula (10).##STR13## Deprotection of allyl ester of Formula (10) usingtriethylsilane in the presence of a catalytic amount oftetrakis(triphenylphosphine)palladium(O) in a suitable solvent such astetrahydrofuran at reflux affords, after acidification with acetic acid,an acid of the Formula (Id), wherein P is CO₂ H. ##STR14## Likewise,compounds of Formula (Ie) can be prepared from isoxazolyl ester ofFormula (6) by reduction with either diisobutylaluminum hydride indichloromethane or lithium borohydride in tetrahydrofuran at lowtemperature to provide the primary alcohol of Formula (11) ##STR15##Oxidation of compound of Formula (11) with Jones reagent at roomtemperature afforded in aldehyde of Formula (12), ##STR16## which wascondensed with half acid of Formula (9), in refluxing benzene and in thepresence of piperidinium acetate with azeotropic removal of water usinga Dean-Stark apparatus, to provide an ester of Formula (13). ##STR17##Deprotection of allyl ester of Formula (13) using triethylsilane in thepresence of a catalytic amount oftetrakis(triphenylphosphine)palladium(O) in a suitable solvent such astetrahydrofuran at reflux affords, after acidification with acetic acid,an acid of the Formula (Ie), wherein P is CO₂ H. ##STR18## Compounds ofFormula (If) can be prepared starting by commercially available ketonesof Formula (14) ##STR19## by reaction with diethyl oxalate of Formula(15) ##STR20## in the presence of a base such as sodium ethoxide in asolvent such as ethanol to produce a diketone of Formula (16). ##STR21##Reaction of a diketone of Formula (16) with hydroxylamine hydrochloride(NH₂ OH.HCl) in a suitable solvent such as pyridine at reflux providesan isoxazole of Formula (17) ##STR22## Reduction of the isoxazol ofFormula (17) with either diisobutylaluminum hydride in dichloromethaneor lithium borohydride in tetrahydrofuran at low temperature provided inthe primary alcohol of Formula (18). ##STR23## Oxidation of theisoxazolyl alcohol of Formula (18) with ether Jones reagent at ambienttemperature or with manganese dioxide in refluxing toluene afforded thealdehyde of Formula (19), ##STR24## which was condensed with half acidof Formula (9) in refluxing benzene in the presence of piperidiniumacetate with azeotropic removal of water to afford an ester of Formula(20). ##STR25## Deprotection of allyl ester of Formula (20) usingtriethylsilane in the presence of a catalytic amount oftetrakis(triphenylphosphine)palladium(O) in a suitable solvent such astetrahydrofuran at reflux affords, after acidification with acetic acid,an acid of the Formula (If), wherein P is CO₂ H and R₁ is H.

In order to use a compound of the Formula (I) or a pharmaceuticallyacceptable salt thereof for the treatment of humans and other mammals itis normally formulated in accordance with standard pharmaceuticalpractice as a pharmaceutical composition.

Compounds of Formula (I) and their pharmaceutically acceptable salts maybe administered in a standard manner for the treatment of the indicateddiseases, for example orally, parenterally, sub-lingually,transdermally, rectally, via inhalation or via buccal administration.

Compounds of Formula (I) and their pharmaceutically acceptable saltswhich are active when given orally can be formulated as syrups, tablets,capsules and lozenges. A syrup formulation will generally consist of asuspension or solution of the compound or salt in a liquid carrier forexample, ethanol, peanut oil, olive oil, glycerine or water with aflavoring or colouring agent. Where the composition is in the form of atablet, any pharmaceutical carrier routinely used for preparing solidformulations may be used. Examples of such carriers include magnesiumstearate, terra alba, talc, gelatin, agar, pectin, acacia, stearic acid,starch, lactose and sucrose. Where the composition is in the form of acapsule, any routine encapsulation is suitable, for example using theaforementioned carriers in a hard gelatin capsule shell. Where thecomposition is in the form of a soft gelatin shell capsule anypharmaceutical carrier routinely used for preparing dispersions orsuspensions may be considered, for example aqueous gums, celluloses,silicates or oils and are incorporated in a soft gelatin capsule shell.

Typical parenteral compositions consist of a solution or suspension ofthe compound or salt in a sterile aqueous or non-aqueous carrieroptically containing a parenterally acceptable oil, for examplepolyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil, orsesame oil.

Typical compositions for inhalation are in the form of a solution,suspension or emulsion that may be administered as a dry powder or inthe form of an aerosol using a conventional propellant such asdichlorodifluoromethane or trichlorofluoromethane.

A typical suppository formulation comprises a compound of Formula (1) ora pharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent, forexample polymeric glycols, gelatins, cocoa-butter or other low meltingvegetable waxes or fats or their synthetic analogues.

Typical transdermal formulations comprise a conventional aqueous ornon-aqueous vehicle, for example a cream, ointment, lotion or paste orare in the form of a medicated plaster, patch or membrane.

Preferably the composition is in unit dosage form, for example a tablet,capsule or metered aerosol dose, so that the patient may administer tothemselves a single dose.

Each dosage unit for oral administration contains suitably from 0.1 mgto 500 mg/Kg, and preferably from 1 mg to 100 mg/Kg, and each dosageunit for parenteral administration contains suitably from 0.1 mg to 100mg, of a compound of Formula (I) or a pharmaceutically acceptable saltthereof calculated as the free acid. Each dosage unit for intranasaladministration contains suitably 1-400 mg and preferably 10 to 200 perperson. A topical formulation contains suitably 0.01 to 1.0% of acompound of Formula (I).

The daily dosage regimen for oral administration is suitably about 0.01mg/Kg to 40 mg/Kg, of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof calculated as the free acid. The daily dosageregimen for parenteral administration is suitably about 0.001 mg/Kg to40 mg/Kg, of a compound of the Formula (I) or a pharmaceuticallyacceptable salt thereof calculated as the free acid. The daily dosageregimen for intranasal administration and oral inhalation is suitablyabout 10 to about 500 mg/person. The active ingredient may beadministered from 1 to 6 times a day, sufficient to exhibit the desiredactivity.

No unacceptable toxicological effects are expected when compounds of theinvention are administered in accordance with the present invention.

The biological activity of the compounds of Formula (I) are demonstratedby the following tests:

I. Binding Assay

A) CHO Cell Membrane Preparation

CHO cells stably transfected with human ET_(A) and ET_(B) receptors weregrown in 245 mm×245 mm tissue culture plates in Dulbecco's modifiedEagle's medium supplemented with 10% fetal bovine serum. The confluentcells were washed with Dulbecco's phosphate-buffered saline containing aprotease inhibitor cocktail (5 mM EDTA, 0.5 ml PMSF, 5 ug/ml ofleupeptin and 0.1 U/ml of aprotinin) and scraped in the same buffer.After centrifugation at 800× g, the cells were lysed by freezing inliquid nitrogen and thawing on ice followed by homogenization (30 timesusing a glass dounce homogenizer) in lysis buffer containing 20 mM TrisHCI, pH 7.5, and the protease inhibitor cocktail. After an initialcentrifugation at 800× g for 10 min to remove unbroken cells and nuclei,the supernatants were centrifuged at 40,000× g for 15 min and the pelletwas resuspended in 50 mM Tris HCI, pH 7.5, and 10 mM MgCl₂ and stored insmall aliquots at -70° C. after freezing in liquid N₂. Protein wasdetermined by using the BCA method and BSA as the standard.

(B) Binding Studies

[¹²⁵ I]ET-1 binding to membranes prepared from CHO cells was performedfollowing the procedure of Elshourbagy et al. (1993). Briefly, the assaywas initiated in a 100 ul volume by adding 25 ul of [¹²⁵ I]ET-1 (0.2-0.3nM) in 0.05% BSA to membranes in the absence (total binding) or presence(nonspecific binding) of 100 nM unlabeled ET-1. The concentrations ofmembrane proteins were 0.5 and 0.05 ug per assay tube for ET_(A) andET_(B) receptors, respectively. The incubations (30° C., 60 min) werestopped by dilution with cold buffer (20 mM Tris HCI, pH 7.6, and 10 mMMgCl₂) and filtering through Whatman GF/C filters (Clifton, N.J.)presoaked in 0.1% BSA. The filters were washed 3 times (5 ml each time)with the same buffer by using a Brandel cell harvester and were countedby using a gamma counter at 75% efficiency.

The following example is illustrative and are not limiting of thecompounds of this invention.

EXAMPLE 1

Formulations for pharmaceutical use incorporating compounds of thepresent invention can be prepared in various forms and with numerousexcipients. Examples of such formulations are given below.

Inhalant Formulation

A compound of Formula I, (1 mg to 100 mg) is aerosolized from a metereddose inhaler to deliver the desired amount of drug per use.

    ______________________________________                                        Tablets/Ingredients                                                                            Per Tablet                                                   ______________________________________                                        1.       Active ingredient                                                                          40 mg                                                      (Cpd of Form. I)                                                             2. Corn Starch  20 mg                                                         3. Alginic acid  20 mg                                                        4. Sodium Alginate  20 mg                                                     5. Mg stearate 1.3 mg                                                           2.3 mg                                                                    ______________________________________                                    

Procedure for Tablets

Step 1 Blend ingredients No. 1, No. 2, No. 3 and No. 4 in a suitablemixer/blender.

Step 2 Add sufficient water portion-wise to the blend from Step 1 withcareful mixing after each addition. Such additions of water and mixinguntil the mass is of a consistency to permit its conversion to wetgranules.

Step 3 The wet mass is converted to granules by passing it through anoscillating granulator using a No. 8 mesh (2.38 mm) screen.

Step 4 The wet granules are then dried in an oven at 140° F. (60° C.)until dry.

Step 5 The dry granules are lubricated with ingredient No. 5.

Step 6 The lubricated granules are compressed on a suitable tabletpress.

Parenteral Formulation

A pharmaceutical composition for parenteral administration is preparedby dissolving an appropriate amount of a compound of formula I inpolyethylene glycol with heating. This solution is then diluted withwater for injections Ph Eur. (to 100 ml). The solution is then steriledby filtration through a 0.22 micron membrane filter and sealed insterile containers.

What is claimed is:
 1. A compound of Formula (I): ##STR26## wherein Z is##STR27## D is O; P is CO₂ H;R^(a) is hydrogen; R₁ is independentlyhydrogen, Ar or C₁₋₆ alkyl; R₂ is Ar, C₁₋₈ alkyl, C(O)R₁₄ or ##STR28##R₃ and R₅ are independently R₁₃ OH, C₁₋₈ alkoxy, S(O)_(q) R₁₁, N(R₆)₂,Br, F, I, Cl, CF₃, NHCOR₆ R₁₃ CO₂ R₇, --X--R₉ Y or --X(CH₂)_(n) R₈wherein each methylene group within --X(CH₂)_(n) R₈ may be unsubstitutedor substituted by one or two --(CH₂)_(n) Ar groups; R₄ is independentlyR₁₁, OH, C₁₋₅ alkoxy, S(O)_(q) R₁₁, N(R₆)₂, Br, F, I, Cl or NHCOR₆,wherein the C₁₋₅ alkoxy may be unsubstituted or substituted by OH,methoxy or halogen; R₆ is independently hydrogen or C₁₋₈ alkyl; R₇ isindependently hydrogen, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₈ alkynyl, allof which may be unsubstituted or substituted by one or more OH, N(R₆)₂,CO₂ R₁₂, halogen or XC₁₋₁₀ alkyl; or R₇ is (CH₂)_(n) Ar; R₈ isindependently R₁₁, CO₂ R₇, CO₂ C(R₁₁)₂ O(CO)XR₇, PO₃ (R₇)₂, SO₂ NR₇ R₁₁,NR₇ SO₂ R₁₁, CONR₇ SO₂ R₁₁, SO₃ R₇, SO₂ R₇, P(O)(OR₇)R₇, CN, CO₂(CH₂)_(m) C(O)N(R₆)₂, C(R₁₁)₂ N(R₇)₂, C(O)N(R₆)₂, NR₇ C(O)NR₇ SO₂ R₁₁,tetrazole or OR₆ ; R₉ is independently a bond, C₁₋₁₀ alkylene, C₁₋₁₀alkenylene, C₁₋₁₀ alkylidene, C₁₋₁₀ alkynylene, all of which may belinear or branched, or phenylene, all of which may be unsubstituted orsubstituted by one of more OH, N(R₆)₂, COOH or halogen; R₁₀ isindependently C₁₋₁₀ alkyl, N(R₆)₂ or Ar; R₁₁ is independently hydrogen,Ar, C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, all of which may beunsubstituted or substituted by one or more OH, CH₂ OH, N(R₆)₂ orhalogen; R₁₂ is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl or C₂₋₇alkynyl; R₁₃ is independently divalent Ar, C₁₋₁₀ alkylene, C₁₋₁₀alkylidene, C₂₋₁₀ alkenylene, all of which may be unsubstituted orsubstituted by one or more OH, CH₂ OH, N(R₆)₂ or halogen; R₁₄ isindependently hydrogen, C₁₋₁₀ alkyl, XC₁₋₁₀ alkyl, Ar or XAr; R₁₅ isindependently C₁₋₆ alkyl or phenyl substituted by one or two C₁₋₆ alkyl,OH, C₁₋₅ alkoxy, S(O)_(q) R₆, N(R₆)₂, Br, F, I, Cl, CF₃ or NHCOR₆ ; X isindependently (CH₂)_(n), O, NR₆ or S(O)_(q) ; Y is independently CH₃ orX(CH₂)_(n) Ar; Ar is independently: ##STR29## naphthyl, furyl, oxozolyl,indolyl, pyridyl, thienyl, oxazolidinyl, thiazolyl, isothiazolyl,pyrazolyl, triazolyl, tetrazolyl, imidazolyl, imidazolidinyl,thiazolidinyl, isoxazolyl, oxadiazolyl, thiadiazolyl, morpholinyl,piperidinyl, piperazinyl, pyrrolyl, or pyrimidyl; all of which may beunsubstituted or substituted by one or more Z₁ or Z₂ groups A isindependently C═O, or (C(R₆)₂)_(m) ; B is independently --CH₂ -- or--O--; Z₁ and Z₂ are independently hydrogen, XR₆, C₁₋₈ alkyl, (CH₂)_(q)CO₂ R₆, C(O)N(R₆)₂, CN, (CH₂)_(n) OH, NO₂, F, Cl, Br, I, N(R₆)₂,NHC(O)R₆, X(CH₂)_(n) R₈, O(CH₂)_(m) C(O)NR_(a) SO₂ R₁₅, (CH₂)_(m)OC(O)NR_(a) SO₂ R₁₅, O(CH₂)_(m) NR_(a) C(O)NR₁ SO₂ R₁₅, or tetrazolylwhich may be substituted or unsubstituted by C₁₋₆ alkyl, CF₃ or C(O)R₆ ;Ar' is pyridyl or thienyl; all of which may be unsubstituted orsubstituted by one or more XR₉ --Y, Z₁ or Z₂ groups; m is independently1 to 3; n is independently 0 to 6; q is independently 0, 1 or 2;provided R₃, R₄ and R₅ are not O--O(CH₂)_(n) Ar; or a pharmaceuticallyacceptable salt thereof.
 2. A compound of claim 1 chosen from the groupconsistingof:(E)-alpha-[[5-[4-[(2-carboxyphenyl)methoxy]-2-methoxypyrid-5-yl]isoxazol-4-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid;(E)-alpha-[[5-(3-carboxy-5-chlorothien-2-yl)isoxazol-4-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid;(E)-alpha-[[3-[4-[(2-carboxyphenyl)methoxy]-2-methoxypyrid-5-yl]isoxazol-4-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid;(E)-alpha-[[3-(3-carboxy-5-chlorothien-2-yl)isoxazol-4-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid;(E)-alpha-[[3-Butyl-4-[4-[(2-carboxyphenyl)methoxy]-2-methoxypyrid-5-yl]isoxazol-5-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid;(E)-alpha-[[5-Butyl-4-[4-[(2-carboxyphenyl)methoxy]-2-methoxypyrid-5-yl]isoxazol-3-yl]methylene]-6-methoxy-1,3-benzodioxole-5-propanoicacid.
 3. A pharmaceutical composition comprising a compound of claim 1and a pharmaceutically acceptable carrier.
 4. A method of treatment ofdiseases caused by an excess of endothelin comprising administering to asubject in need thereof, an effective amount of an endothelin receptorantagonist of claim
 1. 5. A method of treating renal failure,hypertension, pulmonary hypertension or heart failure which comprisesadministering to a subject in need thereof, an effective amount of acompound of claim 1.